We have studied the distribution of the histone variants H3.3 and H2A.Z in chicken erythroid cells. We had shown recently that H3.3 is concentrated at many regulatory regions, as well as over the transcribed regions of some active genes. We have now shown that nucleosomes containing H3.3 are less stable than those containing the predominant species, H3.1, and lose their H2AH2B components more readily. Furthermore, nucleosomes containing H3.3 and H2A.Z are even more unstable. Using double chromatin immunoprecipitation methods we have identified numerous genomic loci at where such doubly substituted nucleosomes are present. They mark transcriptional regulatory sites that control active genes.